Phytoplankton and particle size spectra indicate intense mixotrophic dinoflagellates grazing from summer to winter

Abstract Mixotrophic dinoflagellates (MTD) are a diverse group of organisms often responsible for the formation of harmful algal blooms. However, the development of dinoflagellate blooms and their effects on the plankton community are still not well explored. Here we relate the species succession of MTD with parallel changes of phytoplankton size spectra during periods of MTD dominance. We used FlowCAM analysis to acquire size spectra in the range 2–200 μm every one or two weeks from July to December 2007 at Helgoland Roads (Southern North Sea). Most size spectra of dinoflagellates were bimodal, whereas for other groups, e.g. diatoms and autotrophic flagellates, the spectra were unimodal, which indicates different resource use strategies of autotrophs and mixotrophs. The biomass lost in the size spectrum correlates with the potential grazing pressure of MTD. Based on size-based analysis of trophic linkages, we suggest that mixotrophy, including detritivory, drives species succession and facilitates the formation of bimodal size spectra. Bimodality in particular indicates niche differentiation through grazing of large MTD on smaller MTD. Phagotrophy of larger MTD may exceed one of the smaller MTD since larger prey was more abundant than smaller prey. Under strong light limitation, a usually overlooked refuge strategy may derive from detritivory. The critical role of trophic links of MTD as a central component of the plankton community may guide future observational and theoretical research.


SM2. Validation and limitations of FlowCAM methodology
The acquisition of biomass size spectra is currently impaired by several limitations (Lombard et al. 2019). In our case, the FlowCAM system better captures larger rather than smaller particles (Sieracki et al. 1998). As mentioned in the Material and Methods section, we address this difficulty using two combinations of flowcell+magnification: (1) 100 μm + 20X for small particles (range 2-100 μm) and (2) 300 μm + 10X for larger particles (range 15-300 μm, ).
Further details on the methodology can be found in Hantzsche (2010). From the first combination, separated particle counts were kept for the ranges 2-15 μm and 15-100 μm. From the second combination, particles larger than 200 μm were removed from the dataset due the inverse filtration step conducted using a filter with 250 μm mesh size.
During our study period, the abundance of fluorescent particles in the range 2 to 15 μm were never below 100 cell/mL, reaching a maximum of approx. 2000 cell/mL before and during the dinoflagellate bloom. In comparison, the abundance of particles in the range 15-300 μm was approx. 100 cell/mL, with a maximum of 700 cell/mL during the dinoflagellate bloom ( Fig. SM-9). Despite of the higher abundance of small particles, the chlorophyll concentration was not correlated neither with the particles in the 2-15 μm range nor with the one in the 15-300 μm range. However, particles >50 μm were significantly correlated with chlorophyll concentration (r 2 = 0.67, p < 0.001). From these comparisons we conclude that the biomass share of small cells (<50 μm) is not significant compared to the share of larger cells (>50 μm), at least for chlorophyll containing cells.
As a validation of our methodology, we compared the FlowCAM counts of fluorescent particles with microscopic counts for each size range. The FlowCAM counts of fluorescent particles underestimated the microscopic counts in a factor of approx. 10 for particles in the range 2-15 μm and in approx. 2.5 in the range 15-100 μm for the first flowcell+magnification combination, and in a factor of 2 for the range 15-300 μm for the second combination. We also observed significant correlations between FlowCAM and microscopic counts of flagellates (r 2 = 0.25, p < 0.001) and Akashiwo sanguinea (r 2 = 0.64, p < 0.001). These features were similarly reported for other FlowCAM applications (e.g. Hrycik et al. 2019) and can be summarized as follows: (1) despite the effort of using two different combinations of flowcell+magnification our methodology still presents a sampling bias toward larger cells, and (2) the FlowCAM counts are proportional to the microscopic counts, hence valid to assess the size structure of the phytoplankton community.
We applied two correction factors to the biomass spectra to ameliorate the effects of the sampling bias: 10 for the 2-15 μm fraction and 2 for the 15-300 μm fraction. However, due the small biomass per individual cell in the 2-15 μm fraction these corrections had little effect in our results, which can be noticed in the biomass size-spectra during the entire study period (Fig. SM-7): the bimodality of distributions, the temporal changes in size spectra, as well as the regions of maximum biomass loss are preserved after the bias correction. Bias correction did thus not affect our interpretations.
The little effect of the underestimation of small particles is also observed in the linear regression of the feeding loss index (FLI) as function of the dinoflagellates biomass share (x). The correlation of the feeding loss index with of dinoflagellates biomass share using the sampling bias correction is FLI = 0.47 x while for the uncorrected version is FLI = 0.49 x, both with similar levels of significance (p < 0.001) and the same standard deviation (0.03): the difference of the slopes is not significant (anova test, p>0.1). This result confirms our assumption of a minor influence of the 2-15 μm fraction on trophic dynamics.
The limitations of our method may lead to an underestimation of the phagotrophic activity of small MTD. Small dinoflagellates (P. triestinμm and L. chlorophorum) may in principle graze on small phytoplankton -i.e. flagellates-, especially before and during the onset of the dinoflagellates bloom. However, the low biomass share of flagellates and, in general, the low prey availability for small MTD (Fig. 4b in the main text) possibly renders phagotrophy a highly variable and scarce resource acquisition mechanism. The calculation of prey availability after bias correction does not substantially changed the previously found values (Fig. SM-8). We conclude that the low prey availability for small MTD is a consequence of the biomass size distribution and not an artifact derived by our methodology. Furthermore, we presented the data with no correction factor to enhance the clarity of the presentation of our findings.